Acoustofluidic preparation of whole blood components

Whole blood is rich in information about the physical state of an individual and is routinely used in a variety of clinical and research applications. In this thesis, the use of a microfluidic technique called acoustophoresis for separation of different blood components is explored. Acoustophoresis uses an ultrasonic standing wave field to manipulate cells in a microfluidic device. Cells within the standing wave field will experience forces which move the cells either to the place of minimum pressure, the node, or to the place of maximum pressure, the anti-node. The strength of the acoustic force is dependent on the cells’ properties such as size, density, and compressibility in relation to its surrounding medium. In the first two papers a method is described to use affinity-bead-mediated acoustophoresis toseparate cells which otherwise cannot be acoustically discriminated. Cells of interest were labeled with spherical particles creating large and dense bead-cell complexes which moved faster in the acoustic field compared to unbound cells. The performance was comparable to standard magnetic separation and no effect of the acoustic separation method on viability, cell proliferation or clonogenic stem cell capacity was observed. The third paper investigates the possibility to separate blood cells with similar acoustic mobilities. By changing the properties of the surrounding medium mononuclear cells reduced their mobility in relation to red blood cells and could be successfully separated. The outcomes in terms of efficiency and purity were comparable to conventional separation methods. Next, the possibility to enrich tumor cells from patients undergoing stem cell transplantation was explored. Tumor cells spiked into apheresis products were acoustically separated based on their size differences without interfering with the cells’ viability, T cell activation, and tumor cell proliferation capacity. For the separation outcome a stable temperature is crucial. In the fifth paper a new chip holder was designed to employ an air-cooling unit in order to remove excessiveheat and allow better heat distribution during the separation process. The system allowed high-throughput multiplex separation of particles at flow rates up to 500 μL/min and concurrent separation of the different white blood cell subgroups, i.e. lymphocytes, monocytes, and granulocytes, was achieved for flow rates up to 300 μL/min. Last, to predict the possibility to separate cells in an acoustic standing wave field it is crucial to know the cells properties. Therefore, a method to statistically estimate the cells biomechanical properties based on acoustophoretic separation data was developed. The work presented here shows the diversity, capability and usability of acoustophoresis for whole blood fractionation

High performance multiplex acoustophoresis for WBC subpopulation isolation

Recently, acoustophoresis has been used to fractionate white blood cells (WBC) into subpopulations, Grenvall et al. [1]. However, at a sample throughput of 8-10 μl/min the separation has limited bioanalytical application. In order to substantially increase throughput, we have redesigned and developed a new separation system that enables unmatched WBC separation performance at a volume throughput of 200μl/min and a cell concentration of 106 cells/ml

Microbial pathogen removal from porcine semen with acoustophoresis

Microbial pathogens in semen used for artificial insemination (AI) do not only transmit diseases, but also reduce semen quality. A continuous pathogen removal separation technique overcomes the limits of current processing and separation techniques. We have applied acoustophoresis to separate bacteria and viruses from porcine semen while maintaining spermatozoa viability and achieved high spermatozoa recovery

Label-free separation of leukocyte subpopulations using high throughput multiplex acoustophoresis

Multiplex separation of mixed cell samples is required in a variety of clinical and research applications. Herein, we present an acoustic microchip with multiple outlets and integrated pre-alignment channel to enable high performance and label-free separation of three different cell or particle fractions simultaneously at high sample throughput. By implementing a new cooling system for rigorous temperature control and minimal acoustic energy losses, we were able to operate the system isothermally and sort suspensions of 3, 5 and 7 μm beads with high efficiencies (>95.4%) and purities (>96.3%) at flow rates up to 500 μL min -1 corresponding to a throughput of ∼2.5 × 10 6 beads per min. Also, human viable white blood cells were successfully fractionated into lymphocytes, monocytes and granulocytes with high purities of 96.5 ± 1.6%, 71.8 ± 10.1% and 98.8 ± 0.5%, respectively, as well as high efficiencies (96.8 ± 3.3%, 66.7 ± 3.2% and 99.0 ± 0.7%) at flow rates up to 100 μL min -1 (∼100000 cells per min). By increasing the flow rate up to 300 μL min -1 (∼300000 cells per min) both lymphocytes and granulocytes were still recovered with high purities (92.8 ± 1.9%, 98.2 ± 1.0%), whereas the monocyte purity decreased to 20.9 ± 10.3%. The proposed isothermal multiplex acoustophoresis platform offers efficient fractionation of complex samples in a label-free and continuous manner at thus far unreached high sample throughput rates

Label-free acoustophoretic enrichment of mononuclear cells from blood

We are reporting an efficient, label-free and continuous separation of mononuclear cells (MNC), from blood using microchip acoustophoresis. In standard PBS buffer, MNCs and red blood cells (RBC) display overlapping acoustophoretic mobilities which compromise separation of these cell types from each other. In this paper we capitalize on the fact that MNC and RBC display different acoustophysical properties. By optimizing the buffer conditions and thereby changing the acoustic contrast factor, and hence the acoustophoretic mobility of the cells, a 2800-fold enrichment of MNCs vs. RBCs with MNC recoveries up to 88% was accomplished

Affinity-Bead-Mediated Enrichment of CD8+ Lymphocytes from Peripheral Blood Progenitor Cell Products Using Acoustophoresis

Acoustophoresis is a technique that applies ultrasonic standing wave forces in a microchannel to sort cells depending on their physical properties in relation to the surrounding media. Cell handling and separation for research and clinical applications aims to efficiently separate specific cell populations. Here, we investigated the sorting of CD8 lymphocytes from peripheral blood progenitor cell (PBPC) products by affinity-bead-mediated acoustophoresis. PBPC samples were obtained from healthy donors (n = 4) and patients (n = 18). Mononuclear cells were labeled with anti-CD8-coated magnetic beads and sorted on an acoustophoretic microfluidic device and by standard magnetic cell sorting as a reference method. CD8 lymphocytes were acoustically sorted with a mean purity of 91% ± 8% and a median separation efficiency of 63% (range 15.1%–90.5%) as compared to magnetic sorting (purity 91% ± 14%, recovery 29% (range 5.1%–47.3%)). The viability as well as the proliferation capacity of sorted lymphocytes in the target fraction were unimpaired and, furthermore, hematopoietic progenitor cell assay revealed a preserved clonogenic capacity post-sorting. Bead-mediated acoustophoresis can, therefore, be utilized to efficiently sort less frequent CD8+ lymphocytes from PBPC products in a continuous flow mode while maintaining cell viability and functional capacity of both target and non-target fractions

Label-free neuroblastoma cell separation from hematopoietic progenitor cell products using acoustophoresis - towards cell processing of complex biological samples

Processing of complex cell preparations such as blood and peripheral blood progenitor cell (PBPC) transplants using label-free technologies is challenging. Transplant-contaminating neuroblastoma cells (NBCs) can contribute to relapse, and we therefore aimed to provide proof-of-principle evidence that label-free acoustophoretic separation can be applied for diagnostic NBC enrichment and removal ("purging") from human blood and PBPC products. Neuroblastoma cells spiked into blood and PBPC preparations served as model systems. Acoustophoresis enabled to enrich NBCs from mononuclear peripheral blood cells and PBPC samples with recovery rates of up to 60-97%. When aiming at high purity, NBC purities of up to 90% were realized, however, compromising recovery. Acoustophoretic purging of PBPC products allowed substantial tumour cell depletion of 1.5-2.3 log. PBPC loss under these conditions was considerable (>43%) but could be decreased to less than 10% while still achieving NBC depletion rates of 60-80%. Proliferation of cells was not affected by acoustic separation. These results provide first evidence that NBCs can be acoustically separated from blood and stem cell preparations with high recovery and purity, thus indicating that acoustophoresis is a promising technology for the development of future label-free, non-contact cell processing of complex cell products

Rapid and effective enrichment of mononuclear cells from blood using acoustophoresis

Effective separation methods for fractionating blood components are needed for numerous diagnostic and research applications. This paper presents the use of acoustophoresis, an ultrasound based microfluidic separation technology, for label-free, gentle and continuous separation of mononuclear cells (MNCs) from diluted whole blood. Red blood cells (RBCs) and MNCs behave similar in an acoustic standing wave field, compromising acoustic separation of MNC from RBC in standard buffer systems. However, by optimizing the buffer conditions and thereby changing the acoustophoretic mobility of the cells, we were able to enrich MNCs relative to RBCs by a factor of 2,800 with MNC recoveries up to 88%. The acoustophoretic microchip can perform cell separation at a processing rate of more than 1 × 105 cells/s, corresponding to 5 μl/min undiluted whole blood equivalent. Thus, acoustophoresis can be easily integrated with further down-stream applications such as flow cytometry, making it a superior alternative to existing MNC isolation techniques

Plasma generation and label-free mononuclear cell separation from whole blood by one-step acoustic focusing

We present the first one-step acoustic separation of high quality plasma and mononuclear cells (MNCs) from undiluted human whole blood. This enables centrifugation-free integration in analytical instrumentation

Efficient Purification of CD4+ Lymphocytes from Peripheral Blood Progenitor Cell Products Using Affinity Bead Acoustophoresis

Processing of peripheral blood progenitor cells (PBPC) for clinical transplantation or research applications aims to effectively isolate or deplete specific cell populations, utilizing primarily magnetic or fluorescence activated sorting methods. Here, we investigated the performance of microfluidic acoustophoresis for the separation of lymphocyte subsets from PBPC, and present a novel method for affinity-bead-mediated acoustic separation of cells which can otherwise not be acoustically discriminated. As the acoustic force on a particle depends on particle size, density and compressibility, targeting of cells by affinity specific beads will generate cell-bead complexes that exhibit distinct acoustic properties relative to nontargeted cells and are, thus, possible to isolate. To demonstrate this, PBPC samples (n = 22) were obtained from patients and healthy donors. Following density gradient centrifugation, cells were labeled with anti-CD4-coated magnetic beads (Dynal) and isolated by acoustophoresis and, for comparison, standard magnetic cell sorting technique in parallel. Targeted CD4+ lymphocytes were acoustically isolated with a mean (±SD) purity of 87 ± 12%, compared with 96 ± 3% for control magnetic sorting. Viability of sorted cells was 95 ± 4% (acoustic) and 97 ± 3% (magnetic), respectively. The mean acoustic separation efficiency of CD4+ lymphocytes to the target fraction was 65 ± 22%, compared with a mean CD4+ lymphocyte recovery of 56 ± 15% for magnetic sorting. Functional testing of targeted CD4+ lymphocytes demonstrated unimpaired mitogen-mediated proliferation capacity and cytokine production. Hematopoietic progenitor cell assays revealed a preserved colony forming ability of nontarget cells post sorting. We conclude that the acoustophoresis platform can be utilized to efficiently isolate bead-labeled CD4+ lymphocytes from PBPC samples in a continuous flow format, with preserved functional capacity of both target and nontarget cells. These results open up for simultaneous affinity-bead-mediated separation of multiple cell populations, something which is not possible with current standard magnetic cell separation technolog